ABSTRACT
Telomerase is a ribonucleoprotein (RNP) enzyme that extends telomeric repeats at eukaryotic chromosome ends to counterbalance telomere loss caused by incomplete genome replication. Human telomerase is comprised of two distinct functional lobes tethered by telomerase RNA (hTR): a catalytic core, responsible for DNA extension; and a Hinge and ACA (H/ACA) box RNP, responsible for telomerase biogenesis. H/ACA RNPs also have a general role in pseudouridylation of spliceosomal and ribosomal RNAs, which is critical for the biogenesis of the spliceosome and ribosome. Much of our structural understanding of eukaryotic H/ACA RNPs comes from structures of the human telomerase H/ACA RNP. Here we report a 2.7 Å cryo-electron microscopy structure of the telomerase H/ACA RNP. The significant improvement in resolution over previous 3.3 Å to 8.2 Å structures allows us to uncover new molecular interactions within the H/ACA RNP. Many disease mutations are mapped to these interaction sites. The structure also reveals unprecedented insights into a region critical for pseudouridylation in canonical H/ACA RNPs. Together, our work advances understanding of telomerase-related disease mutations and the mechanism of pseudouridylation by eukaryotic H/ACA RNPs.
Subject(s)
Ribonucleoproteins , Telomerase , Humans , Ribonucleoproteins/genetics , Telomerase/genetics , Cryoelectron Microscopy , Ribonucleoproteins, Small Nucleolar/genetics , RNA/genetics , RNA, RibosomalABSTRACT
Telomerase maintains genome stability by extending the 3' telomeric repeats at eukaryotic chromosome ends, thereby counterbalancing progressive loss caused by incomplete genome replication. In mammals, telomerase recruitment to telomeres is mediated by TPP1, which assembles as a heterodimer with POT1. We report structures of DNA-bound telomerase in complex with TPP1 and with TPP1-POT1 at 3.2- and 3.9-angstrom resolution, respectively. Our structures define interactions between telomerase and TPP1-POT1 that are crucial for telomerase recruitment to telomeres. The presence of TPP1-POT1 stabilizes the DNA, revealing an unexpected path by which DNA exits the telomerase active site and a DNA anchor site on telomerase that is important for telomerase processivity. Our findings rationalize extensive prior genetic and biochemical findings and provide a framework for future mechanistic work on telomerase regulation.
Subject(s)
DNA/chemistry , Shelterin Complex/chemistry , Telomerase/chemistry , Telomere-Binding Proteins/chemistry , Telomere/metabolism , Amino Acid Motifs , Catalytic Domain , Cryoelectron Microscopy , DNA/metabolism , Humans , Models, Molecular , Protein Binding , Protein Conformation , Protein Domains , Protein Interaction Domains and Motifs , Shelterin Complex/metabolism , Telomere-Binding Proteins/metabolismABSTRACT
The IL-33-ST2 pathway is an important initiator of type 2 immune responses. We previously characterised the HpARI protein secreted by the model intestinal nematode Heligmosomoides polygyrus, which binds and blocks IL-33. Here, we identify H. polygyrus Binds Alarmin Receptor and Inhibits (HpBARI) and HpBARI_Hom2, both of which consist of complement control protein (CCP) domains, similarly to the immunomodulatory HpARI and Hp-TGM proteins. HpBARI binds murine ST2, inhibiting cell surface detection of ST2, preventing IL-33-ST2 interactions, and inhibiting IL-33 responses in vitro and in an in vivo mouse model of asthma. In H. polygyrus infection, ST2 detection is abrogated in the peritoneal cavity and lung, consistent with systemic effects of HpBARI. HpBARI_Hom2 also binds human ST2 with high affinity, and effectively blocks human PBMC responses to IL-33. Thus, we show that H. polygyrus blocks the IL-33 pathway via both HpARI which blocks the cytokine, and also HpBARI which blocks the receptor.
